Biopharmconsortium Blog

Expert commentary from Haberman Associates biotechnology and pharmaceutical consulting.

Posts filed under: Biomarkers

Can adoptive cellular immunotherapy successfully treat metastatic gastrointestinal cancers?

 

Dr. Steven Rosenberg

Dr. Steven Rosenberg

On September 6, 2014, we published an article on this blog announcing the publication of our book-length report, Cancer Immunotherapy: Immune Checkpoint Inhibitors, Cancer Vaccines, and Adoptive T-cell Therapies, by Cambridge Healthtech Institute (CHI).

In that article, we cited the example of the case of a woman with metastatic cholangiocarcinoma (bile-duct cancer), which typically kills the patient in a matter of months. The patient, Melinda Bachini, was treated via adoptive immunotherapy with autologous tumor-infiltrating T cells (TILs) resulting in survival over a period of several years, with a good quality of life.

Our report includes a full discussion of that case, as of the date of the May 2014 publication of a report in Science by Steven A. Rosenberg, M.D., Ph.D. and his colleagues at the National Cancer Institute (NCI). Ms. Bachini’s story was also covered in a May 2014 New York Times article.

Now comes the publication, in Science on December 2015, of an update from the Rosenberg group on their clinical studies of TIL-based immunotherapy of metastatic gastrointestinal cancers. This article discusses the results of TIL treatment of ten patients with a variety of gastrointestinal cancers, including cancers of the bile duct, the colon or rectum, the esophagus, and the pancreas. The case of Ms. Bachini (“patient number 3737”) was included.

Ms. Bachini, a paramedic and a married mother of six children, and a volunteer with the Cholangiocarcinoma Foundation, was 41 years old when first diagnosed with cancer. She remains alive today—a five-year survivor—at age 46.

The Foundation produced a video, dated March 13, 2015, in which Ms. Bachini gives her “patient perspective”. This video includes her story “from the beginning”—from diagnosis through surgery and chemotherapy, and continuing with adoptive immunotherapy at the NCI under Dr. Rosenberg. Although her tumors continue to shrink and she remains alive, she still is considered to have “Stage 4” (metastatic) cancer. Ms. Bachini is a remarkable woman.

The Cholangiocarcinoma Foundation has also produced an on-demand webinar (dated October 21, 2014) on the adoptive cellular therapy trial in patients with various types of metastatic gastrointestinal cancers, led by Drs. Eric Tran and Steven Rosenberg. Ms. Bachini is also a presenter on that webinar. The December 2015 Science article is an updated version of the results of this trial.

The trial, a Phase 2 clinical study (NCT01174121) remains ongoing, and is recruiting new patients.

The particular focus of Dr. Tran’s and Dr. Rosenberg’s study in TIL treatment of gastrointestinal cancers is whether TILs derived from these tumors include T-cell subpopulations that target specific somatic mutations expressed by the cancers, and whether these subpopulations might be harnessed to successfully treat patients with these cancers. Of the ten patients who were the focus of the December 2015 publication, only Ms. Bachini had a successful treatment. In the case of Ms. Bachini, she received a second infusion of TILs that were enriched for CD4+ T cells that targeted a unique mutation in a protein known as ERBB2IP. It was this second treatment that resulted in the successful knockdown of her tumors, which continues to this day.

Despite the lack of similar successes in the treatment of the other nine patients, the researchers found that TILs from eight of these patients contained CD4+ and/or CD8+ T cells that recognized one to three somatic mutations in the patient’s own tumors. Notably, CD8+ TILs isolated from a colon cancer tumor of one patient (patient number 3995) recognized a mutation in KRAS known as KRAS G12D. This mutation results in an amino acid substitution at position 12 in KRAS, from glycine (G) to aspartic acid (D). KRAS G12D is a driver mutation that is involved in causation of many human cancers.

Although two other patients (numbers 4032 and 4069, with colon and pancreatic cancer, respectively) had tumors that expressed KRAS G12D, the researchers did not detect TILs that recognized the KRAS mutation in these patients. The researchers concluded that KRAS G12D was not immunogenic in these patients. The TILs from patient 3995 were CD8+ T cells that recognized KRAS G12D in the context of the human leukocyte antigen (HLA) allele HLA-C*08:02. [As with all T cells, TILs express T-cell receptors (TCRs) that recognize a specific antigenic peptide bound to a particular major histocompatibility complex (MHC) molecule—this is referred to as “MHC restriction”.] The two patients for whom KRAS G12D was not immunogenic did not express the HLA-C*08:02 allele.

The results seen with KRAS G12D-expressing tumor suggest the possibility of constructing genetically-engineered CD8+ T cells that express a TCR that is reactive with the KRAS mutation in the context of the HLA-C*08:02 allele. The KRAS G12D driver mutation is expressed in about 45% of pancreatic adenocarcinomas, 13% of colorectal cancers, and at lower frequencies in other cancers, and the HLA-C*08:02 allele is expressed by approximately 8% and 11% of white and black people, respectively, in the U.S. Thus, in the U.S. alone, thousands of patients per year with metastatic gastrointestinal cancers would potentially be eligible for immunotherapy with this KRASG12D-reactive T cell.

Although only Ms. Bachini (“patient number 3737”) was a long-term survivor, the researchers were able to treat three other patients with enriched populations of TILs targeting predominantly one mutated tumor antigen. Patient 4069 experienced a transient regression of multiple lung metastases of his pancreatic adenocarcinoma, but patients 4007 and 4032 had no objective response. Whereas 23% of circulating T cells at one month after treatment were adoptively transferred mutation-specific TILs in the case of Ms. Bachini, the other three patients treated with enriched populations of mutation-specific TILs showed no or minimal persistence. The researchers concluded that they will need to develop strategies designed to enhance the potency and persistence of adoptively transferred mutation-specific TILs. Nevertheless, the researchers concluded that nearly all patients with advanced gastrointestinal cancers harbor tumor mutation-specific TILs. This finding may serve as the basis for developing personalized adoptive cellular therapies and/or vaccines that can effectively target common epithelial cancers.

Conclusions

Dr. Rosenberg pioneered the study and development of adoptive cellular immunotherapy, beginning in the 1980s. Most studies with TIL-based adoptive immunotherapy have been in advanced melanoma. Adoptive cellular immunotherapy is the most effective approach to inducing complete durable regressions in patients with metastatic melanoma.

As we discussed in our cancer immunotherapy report, melanoma tumors have many more somatic mutations (about 200 nonsynonymous mutations per tumor) than most types of cancer. This appears to be due to the role of a potent immunogen—ultraviolet light—in the pathogenesis of melanoma. The large number of somatic mutations in melanomas results in the infiltration of these tumors by TILs that target the mutations. As discussed in our report, Dr. Rosenberg and his colleagues cultured TIL cell lines that addressed specific immunodominant mutations in patients’ melanomas. Treatment with these cell lines in several cases resulted in durable complete remissions of the patients’ cancers.

Dr. Rosenberg and his colleagues used the same strategy employed in identification of TIL cell lines that targeted specific mutations in melanomas to carry out the study in gastrointestinal cancers, as discussed in our report. However, the small number of somatic mutations and of endogenous TILs in gastrointestinal cancers and in most other epithelial cancers has made studies in these cancers more difficult than studies in melanoma.

in addition, the susceptibility of melanoma to treatment with checkpoint inhibitors such as the PD-1 blockers pembrolizumab (Merck’s Keytruda) and nivolumab (Bristol-Myers Squibb’s Opdivo) correlates with the large number of somatic mutations in this type of cancer. As we discussed in our December 15, 2014 article on this blog, immune checkpoint inhibitors work by reactivating endogenous tumor-infiltrating T cells (TILs). In the case of melanoma, these endogenous TILs target the numerous somatic mutations found in these cancers, and—as suggested by Dr. Rosenberg’s studies with cultured TIL cell lines—those endogenous TILs that target immunodominant mutations can induce durable compete remissions. As discussed in our December 15, 2014 blog article, the three major types of immuno-oncology treatments—immune checkpoint inhibitors, cancer vaccines, and adoptive T-cell therapies, work via related mechanisms.

In 2015, researchers showed that other types of cancers that have numerous somatic mutations are especially susceptible to checkpoint inhibitor treatment. These include, for example, non-small cell lung cancers (NSCLCs) that have mutational signatures that indicate that the cancers were caused by smoking, and cancers that have mutations in genes involved in DNA repair. (Mutations in genes involved in DNA repair pathways result in the generation of numerous additional mutations.)

Moreover, as discussed in our December 15, 2014 blog article, cancer immunotherapy researchers have been expanding the types of tumors that can be treated with checkpoint inhibitors. Genentech/Roche’s PD-L1 inhibitor that was discussed in that article, MPDL3280A, is now called atezolizumab. The clinical trials of atezolizumab discussed in that article and in our report have continued to progress. In a pivotal Phase 2 study in locally advanced or metastatic urothelial bladder cancer (UBC), atezolizumab shrank tumors in 27 percent of people whose disease had medium and high levels of PD-L1 expression and had worsened after initial treatment with platinum chemotherapy. These responses were found to be durable. According to Genentech, these results may represent the first major treatment advance in advanced UBC in nearly 30 years. Atezolizumab also gave positive results in Phase 2 clinical trials in patients with NSCLC that expresses medium to high levels of PD-L1.

Meanwhile, NewLink Genetics (Ames, IA) has entered Phase 3 clinical trials in pancreatic cancer with its HyperAcute cellular immunotherapy vaccine therapy. A Phase 2 trial of the company’s HyperAcute cellular immunotherapy algenpantucel-L in combination with chemotherapy and chemoradiotherapy in resected pancreatic cancer (clinical trial number NCT00569387) appears to be promising.

Dr. Rosenberg’s studies of TIL therapies of gastrointestinal cancers represent another approach to moving immuno-oncology treatments beyond melanoma, based on mutation-specific targeting. The types of cancers that form the focus of these studies—gastrointestinal epithelial cancers—have proven difficult to treat. Moreover, several of them are among the most common of cancers. The researchers and patients involved in these and other immuno-oncology studies are heroes, and oncologists appear to be making measured progress against cancers that have been until recently considered untreatable.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to your company, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Immune checkpoint inhibitors work by reactivating tumor-infiltrating T cells (TILs)

cancer cell

Cancer Cell

The 27 November issue of Nature contains a wealth of new studies on how immune checkpoint inhibitors target various types of cancer, and how researchers and physicians might be able to identify the patients who are most likely to benefit from treatment with these agents.

These studies are described in five papers published in that issue of Nature. This issue also contains a “News & Views” commentary on these articles by Drs. Jedd D. Wolchok and Timothy A. Chan (both at the Memorial Sloan Kettering Cancer Center). This article serves as an introduction to the five research articles.

In addition, Science Magazine published a commentary on these articles, entitled “Multiple boosts for cancer immunotherapy”, by contributing correspondent Mitch Leslie.

Checkpoint inhibitors can be used to treat several types of cancer

One important result of these studies is the expansion of the range of cancers that can be treated via immunotherapy beyond melanoma, kidney cancer, and non-small cell lung cancer (NSCLC). The papers by Powles et al. and Herbst et al. contain results from a Phase 1 clinical trial of Genentech’s monoclonal antibody (MAb) PD-L1 blocker MPDL3280A. Herbst et al. reported that MPDL3280A showed therapeutic responses in patients with NSCLC, melanoma, renal cancer, and head and neck cancer. Powles et al. focused on the effects of this agent in a larger group of patients with metastatic urothelial bladder cancer (UBC). In both reports, researchers documented that a subset of patients experienced durable responses, and that the treatment showed low toxicity.

We discussed earlier presentations of the results of the Phase 1 trial of MPDL3280A in our Insight Pharma Report (IPR), Cancer Immunotherapy: Immune Checkpoint Inhibitors, Cancer Vaccines, and Adoptive T-Cell Therapies. As we discussed in this report, the FDA granted breakthrough therapy designation for MPDL3280A for treatment of UBC. Roche/Genentech has initiated a Phase 2 clinical trial (clinical trial number NCT02108652) of MPDL3280A in UBC. UBC is the ninth most common cancer in the world. Metastatic UBC is associated with a poor prognosis, and has few treatment options. There have been no new treatment advances in nearly 30 years.

Checkpoint inhibitors work by reactivating tumor-infiltrating T cells (TILs)

Perhaps the most important finding of the research published in the November 27th issue of Nature is that checkpoint inhibitors work via reactivating endogenous tumor-infiltrating T cells. (These T cells are often called “TILs”, which is an acronym for “tumor-infiltrating lymphocytes”.)

For example, as described in the Powles et al. report, Genentech’s PD-L1 blocker MPDL3280A was found to be especially effective in treating patients whose tumors contained PD-L1-positive TILs. As we discussed in our IPR report, Genentech researchers found that MPDL3280A not only targets PD-L1 on the surface of tumor cells, but also PD-L1 on the surface of TILs. PD-L1 on activated T cells interacts not only with PD-1, but also with B7 on the surface of antigen presenting cells, sending a negative signal to the T cells. MPDL3280A targets the PD-L1-B7 interaction, thus enabling reactivation of PD-L1-bearing TILs so that they can attack the tumor.

As we also discuss in our report, targeting PD-1, PD-L1, and CTLA-4 may also be important in reversing immunosuppression by regulatory T cells (Tregs), which typically heavily infiltrate tumors. This provides another mechanism by which checkpoint inhibitors can reactivate TILs and thus induce anti-tumor immune responses.

As described in Powles et al, MPDL3280A was engineered with a modification in the Fc domain that eliminates antibody-dependent cellular cytotoxicity (ADCC). Genentech researchers did this because PD-L1 is expressed on activated T cells, and they wanted an anti-PD-L1 MAb agent that would reactivate these T cells, not destroy them via ADCC.

In the studies described by Herbst et al., researchers showed that Genentech’s PD-L1 blocker MPDL3280A gives antitumor response across multiple types of cancer, in tumors that expressed high levels of PD-L1. These responses especially occurred when PD-L1 was expressed by TILs. The studies suggest that MPDL3280A is most effective against tumors in which endogenous TILs are suppressed by PD-L1, and are reactivated via anti-PD-L1 MAb targeting.

In the Tumeh et al. study, the researchers found that patients responding to treatment with Merck’s MAb PD-1 blocker pembrolizumab (Keytruda) showed proliferation of intratumoral CD8+ T cells that correlated with reduction in tumor size. Pretreatment tumor samples taken from responding patients showed higher numbers of CD8, PD-1, and PD-L1 expressing cells at the invasive tumor margin and within tumors, with a close proximity between PD-1 and PD-L1, and a clonal TCR repertoire.

Based on this information, the researchers developed a predictive model based on CD8 expression at the invasive tumor margin. They validated this model in an independent 15-patient cohort. The researchers concluded that tumor regression due to treatment with the PD-1 blocker pembrolizumab requires preexisting CD8+ T cells whose activity has been blocked by PD-1/PD-L1 adaptive resistance. This study, like those of Powles et al. and Herbst et al., thus indicate that checkpoint inhibitors work against cancer by reactivating TILs. The Tumeh et al. study also indicates that CD8 expression at the invasive tumor margin is a predictive biomarker for sensitivity of patient tumors to treatment with anti-PD-1 checkpoint inhibitors.

The Powles, Herbst, and Tumeh reports all involved studies in human patients. However, the other two papers—Yadav et al. and Gubin et al. involve studies in mouse tumor models.

In the study of Yadav et al., the researchers used their mouse model to develop a method for discovering immunogenic mutant peptides in cancer cells that can serve as targets for T cells. They sequenced the exomes of two mouse cancer cell lines, and looked for differences with the corresponding normal mouse exomes. They also identified which of the neoantigens that they identified via exome sequencing could bind to histocompatibility complex class I (MHCI) proteins, and thus could be presented to T cells. They then modeled the MHC1/peptide complexes, and used these models to predict which of these neoantigens were likely to be immunogenic.

These methods identified only a few candidate neoantigens. Vaccination of tumor-bearing mice with these neoantigens resulted in therapeutically active T-cell responses. In addition, the researchers developed methods for monitoring the antitumor T cell response to peptide vaccination.

In the study of Gubin et al., the researchers used similar genomic and bioinformatic approaches to those of Yadav et al., and identified two neoantigens that were targeted by T cells following therapy with anti-PD-1 and/or anti-CTLA-4 antibodies. [Human CTLA-4 is the target of the checkpoint blockade inhibitor ipilimumab (Medarex/ Bristol-Myers Squibb’s Yervoy).] As with PD-1 and PD-L1 blockers, we discussed this agent in our IPR report. T cells specific for these neoantigens (in the context of MHCI proteins expressed by the mice) were present in the tumors. These T cells were reactivated by anti-PD-1 and/or anti-CTLA-4 antibodies, enabling the mice to reject the tumors.

As in the study of Yadav et al., the Gubin et al. researchers performed experiments in which they vaccinated tumor-bearing mice with peptides that incorporated the mutant epitopes. This vaccination induced specific tumor rejection that was comparable to treatment with checkpoint blockade inhibitors. As in the case of Yadav et al, the Gubin et al. researchers concluded that specific mutant antigens were targets of checkpoint inhibitor therapy in their mouse models, and that the mutant antigens could also be used to develop personalized cancer vaccines.

Since the studies of Yadav et al. and Gubin et al. were carried out using mouse tumor models, the results are not directly applicable to cancer in human patients. However, the studies suggest that immune checkpoint inhibitors work by reactivating endogenous TILs, and that anti tumor TILs work by attacking specific neoantigens on the tumors.

As we discussed in our IPR report, Dr. Steven Rosenberg (National Cancer Institute, Bethesda, MD) identified specific antigens that were the targets of TILs, both in metastatic melanoma and in metastatic cholangiocarcinoma (a type of epithelial bile duct cancer). However, these target antigens were from human cancers, and they were targets of TILs that has been isolated from patient tumors, cultured and expanded ex vivo, and used in adoptive cellular immunotherapy.

Moreover, the antigens were targets of TIL therapies that resulted in a durable compete remission in the case of the melanoma patient, and long-term tumor regression in the case of the metastatic cholangiocarcinoma patient. The metastatic cholangiocarcinoma case was highlighted in our September 16, 2014 Biopharmconsortium Blog article.

The Yadav et al. paper referenced the Rosenberg group’s work. However, this paper stated that “few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing.”

The methods of Yadav et al. (and of Gubin et al.) are thus designed to simplify and accelerate the discovery of immunogenic mutant peptides. They carried out their studies in mouse models, which helped these researchers to develop methods that could potentially discover greater numbers of neoantigens more efficiently. However, it remains to be seen to what extent they can apply their methods to human patients.

Unifying the field of immuno-oncology

As can be seen, for example, from the title of our IPR report, the three major approaches to immuno-oncology in 2014/2015 are development of immune checkpoint inhibitors, of cancer vaccines, and of adoptive T-cell therapies.

In the immuno-oncology papers published in the 27 November issue of Nature, researchers show that checkpoint inhibitors work via reactivating of endogenous TILs. They also (in mouse tumor models) identified neoantigens that are targets of these reactivated TILs, and designed peptide vaccines that were as effective as checkpoint inhibitor therapy in the mouse models. In principle, one can isolate TILs that are reactive to particular neoantigens in the mouse tumors, culture and expand them ex vivo, and infuse them back into the mice to target their tumors. Thus the studies in the 27 November issue of Nature serve as a template for the unification of the immuno-oncology field as it now exists.

However, it will be necessary to apply the methodologies developed by Yadav et al. and Gubin et al. to human patients. And at least so far, peptide vaccines have not been very successful in treating patients, as compared to TIL therapy (in the subset of patients in whom TIL therapy can be done). It is thus possible that once these methods of neoantigen identification are applied to human patients, it will be found that targeting the neoantigens with ex vivo-expanded TILs will be more successful than therapy with peptide vaccines. However, whether this is true awaits the application of the new methodologies to neoantigen identification in human tumors.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to your company, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Chemokine receptors and the HIV-1 entry inhibitor maraviroc

Maraviroc

Maraviroc

In April 2012, Informa’s Scrip Insights published our book-length report, “Advances in the Discovery of Protein-Protein Interaction Modulators.” We also published a brief introduction to this report, highlighting the strategic importance of protein-protein interaction (PPI) modulators for the pharmaceutical industry, on the Biopharmconsortium Blog.

The report included a discussion on discovery and development of inhibitors of chemokine receptors. Chemokine receptors are members of the G-protein coupled receptor (GPCR) superfamily. GPCRs are seven-transmembrane (7TM) domain receptors (i.e. integral membrane proteins that have seven membrane-spanning domains). Compounds that target GPCRs represent the largest class of drugs produced by the pharmaceutical industry. However, in the vast majority of cases, these compounds target GPCRs that bind to natural small-molecule ligands.

Chemokine receptors, however, bind to small proteins, the chemokines. These proteins constitute a class of small cytokines that guide the migration of immune cells via chemotaxis. Chemokine receptors are thus a class of GPCRs that function by forming PPIs. Direct targeting of interactions between chemokines and their receptors (unlike targeting the interactions between small-molecule GPCR ligands and their receptors) thus involves all the difficulties of targeting other types of PPIs.

However, GPCRs–including chemokine receptors–appear to be especially susceptible to targeting via allosteric modulators. Allosteric sites lie outside the binding site for the protein’s natural ligand. However, modulators that bind to allosteric sites change the conformation of the protein in such a way that it affects the activity of the ligand binding site. (Direct GPCR modulators that bind to the same site as the GPCR’s natural ligands are known as orthosteric modulators.) In the case of chemokine receptors, researchers can in some cases discover small-molecule allosteric modulators that activate or inhibit binding of the receptor to its natural ligands. Discovery of such allosteric activators is much easier than discovery of direct PPI modulators.

Chemokines bind to sites that are located in the extracellular domains of their receptors. Allosteric sites on chemokine receptors, however, are typically located in transmembrane domains that are distinct from the chemokine binding sites. Small-molecule allosteric modulators that bind to these sites were discovered via fairly standard medicinal chemistry and high-throughput screening, sometimes augmented with structure-based drug design. This is in contrast to attempts to discover small molecule agents that directly inhibit binding of a chemokine to its receptor, which has so far been extremely challenging.

Our report describes several allosteric chemokine receptor modulators that are in clinical development, as well as the two agents that have reached the market. One of the marketed agents, plerixafor (AMD3100) (Genzyme’s Mozobil), is an inhibitor of the chemokine receptor CXCR4. It is used in combination with granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells to the peripheral blood for autologous transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. The other agent, which is the focus of this blog post, is maraviroc (Pfizer’s Selzentry/Celsentri).

Maraviroc is a human immunodeficiency virus-1 (HIV-1) entry inhibitor. This compound is an antagonist of the CCR5 chemokine receptor. CCR5 is specific for the chemokines RANTES (Regulated on Activation, Normal T Expressed and Secreted) and macrophage inflammatory protein (MIP) 1α and 1β.  In addition to being bound and activated by these chemokines, CCR5 is a coreceptor (together with CD4) for entry of the most common strain of HIV-1 into T cells. Thus maraviroc acts as an HIV entry inhibitor; this is the drug’s approved indication in the U.S. and in Europe. Maraviroc was discovered via a combination of high-throughput screening and optimization via standard medicinal chemistry.

New structural biology studies of the CCR5-maraviroc complex

Now comes a report in the 20 September 2013 issue of Science on the structure of the CCR5-maraviroc complex. This report was authored by a mainly Chinese group led by Beili Wu, Ph.D. (Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai); researchers at the University of California at San Diego and the Scripps Research Institute, San Diego were also included in this collaboration. A companion Perspective in the same issue of Science was authored by P. J. Klasse, M.D., Ph.D. (Weill Cornell Medical College, Cornell University, New York, NY).

As described in the Perspective, the outer surface of the HIV-1 virus displays numerous envelope protein (Env) trimers, each including the outer gp120 subunit anchored in the viral membrane by gp41. When gp120 binds to the cell-surface receptor CD4, this enables interaction with a specific chemokine receptor, either CCR5 or CXCR4. Interaction with both CD4 and the chemokine receptor triggers complex sets of changes in the Env complex, eventually resulting in the fusion of the viral membrane and the cell membrane, and the entry of the virus particle into the host cell.

HIV-1 gp120 makes contact with CCR5 at several points. The interactions between CCR5 and the variable region of gp120 called V3 are especially important for the tropism of an HIV-1 strain, i.e., whether the virus is specific for CCR5 (the “R5 phenotype”) or CXCR4 (the “X4 phenotype”). In the case of R5-tropic viruses, the tip of the V3 region interacts with the second extracellular loop (ECL2) of CCR5, while the base of V3 interacts with the amino-terminal segment of CCR5. Modeling of the interactions between the V3 domain of gp120 of either R5 or X4-tropic viruses with CCR5 or CXCR4 explains coreceptor use, in terms of forming strong bonds or–conversely–weak bonds and steric hindrance.

Monogram Biosciences (South San Francisco, CA) has developed and markets the Trofile assay. This is a molecular assay designed to identify the R5, X4, or mixed tropism of a patient’s HIV strain. If a patient’s strain is R5-tropic, then treatment with maraviroc is appropriate. However, a patient’s HIV-1 strain may undergo a tropism switch, or may mutate in other ways to become resistant to maraviroc.

Dr Wu and her colleagues determined the high-resolution crystal structure of the complex between maraviroc and a solubilized engineered form of CCR5. This included determining the CCR5 binding pocket for maraviroc, which was determined both by Wu et al’s X-ray crystallography, and by site-directed mutagenesis (i.e., to determine amino acid residues that are critical for maraviroc binding) that had been published earlier by other researchers.

The structural studies of Dr. Wu and her colleagues show that the maraviroc-binding site is different from the recognition sites for gp120 and for chemokines, as expected for an allosteric inhibitor. The X-ray structure shows that maraviroc binding prevents the helix movements that are necessary for binding of g120 to induce the complex sequence of changes that result in fusion between the viral and cellular membranes. (These helix movements are also necessary for induction of signal transduction by binding of chemokines to CCR5.)

Structural studies of CXCR4 and its inhibitor binding sites

In addition to their structural studies of the CCR5-maraviroc complex, Dr. Wu and her colleagues also published structural studies of CXCR4 complexed with small-molecule and cyclic peptide inhibitors in Science in 2010. These inhibitors are IT1t, a drug-like orally-available isothiourea developed by Novartis, and CVX15, a 16-residue cyclic peptide that had been previously characterized as an HIV-inhibiting agent. IT1t and CVX15 bind to overlapping sites in CXCR4. Other researchers have found evidence that the binding site for plerixafor also overlaps with the IT1t binding site.

As discussed in Wu et al’s 2013 paper, CCR5 and CXCR4 have similar, but non-identical structures. The binding site for IT1t in CXCR4 is closer to the extracellular surface than is the maraviroc binding site in CCR5, which is deep within the CCR5 molecule. The entrance to the CXCR4 ligand-binding pocket is partially covered by CXC4’s N terminus and ECL2, but the CCR5 ligand-binding pocket is more open.

Mechanisms of CXCR4 and CCR5 inhibition, and implications for discovery of improved HIV entry inhibitors

The chemokine that specifically interacts with the CXCR4 receptor is known as CXCL12 or stromal cell-derived factor 1 (SDF-1). Researchers have proposed a hypothesis for how CXCL12 interacts with CXCR4; this hypothesis appears to be applicable to the interaction between other chemokines and their receptors as well. This hypothesis is know as the “two-step model” or the “two-site model” of chemokine-receptor activation. Under the two-site model, the core domain of a chemokine binds to a site on its receptor (known as the “chemokine recognition site 1” or “site 1”) defined by the receptor’s N-terminus and its ECLs. In the second step, the flexible N-terminus of the chemokine interacts with a second site (known as “chemokine recognition site 2” or “site 2” or the “activation domain”) deeper within the receptor, in transmembrane domains. This result in activation of the chemokine receptor and intracellular signaling.

Under the two-site model, CXCR4 inhibitors (e.g., IT1t, CVX15, and  plerixafor), which bind to sites within the ECLs of CXCR4, are competitive inhibitors of binding of the core domain of CXCL12 to CXCR4 (i.e.., step 1 of chemokine/receptor interaction). They are thus orthosteric inhibitors of CXCR4. (This is contrary to the earlier assignment of plerixafor as an allosteric inhibitor of CXCR4.)  The CCR5 ligand maraviroc, however, binds within a site within the transmembrane domains of CCR5, which overlaps with the activation domain of CCR5. Dr. Wu and her colleagues propose two alternative hypotheses: 1. Maraviroc may inhibit CCR5 activation by chemokines by blocking the second step of chemokine/chemokine receptor interaction, i.e., receptor activation. 2. Maraviroc may stabilize CCR5 in an inactive conformation. It is also possible that maraviroc inhibition of CCR5 may work via both mechanisms.

Dr. Wu and her colleagues further hypothesize that the interaction of  HIV-1 gp120 with CCR5 (or CXCR4) may operate via similar mechanisms to the interaction of chemokines with their receptors. As we discussed earlier in this article, the base (or the stem region) of the gp120 V3 domain interacts with the amino-terminal segment of CCR5. The tip (or crown) of the V3 domain interacts with the ECL2 of CCR5, and–according to Dr. Wu and her colleagues–also with amino acid residues inside the ligand binding pocket; i.e., the activation site of CCR5. The HIV gp120 V3 domain may thus activate CCR5 via a similar mechanism to the two-step  model utilized by chemokines.

Based on their structural biology studies, Dr. Wu and her colleagues have been building models of the CCR5-R5-V3 and CXC4-X4-V3 complexes, and are also planning to determine additional structures needed to fully understand the mechanisms of HIV-1 tropism. The researchers will utilize their studies in the discovery of improved, second-generation HIV entry inhibitors for both R5-tropic and X4-tropic strains of HIV-1.

The bigger picture

The 17 October 2013 issue of Nature contains a Supplement entitled “Chemistry Masterclass”. In that Supplement is an Outlook review entitled “Structure-led design”, by Nature Publishing Group Senior Editor Monica Hoyos Flight, Ph.D. The subject of this article is structure-based drug design of modulators of GPCRs.
This review outlines progress in determining GPCR structures, and in using this information for discovery of orthosteric and allosteric modulators of GPCRs.

According to the article, the number of solved GPCR structures has been increasing since 2008, largely due to the efforts of the Scripps GPCR Network, which was established in that year. Dr. Wu started her research on CXCR4 and CCR5 as a postdoctoral researcher in the laboratory of Raymond C. Stevens, Ph.D. at Scripps in 2007, and continues to be a member of the network. The network is a collaboration that involves over a dozen academic and industrial labs. Its goal has been to characterize at least 15 GPCRs by 2015; it has already solved 13.

Interestingly, among the solved GPCR structures are those for the corticotropin-releasing hormone receptor and the glucagon receptor. Both have peptide ligands, and thus work by forming PPIs.

One company mentioned in the article, Heptares Therapeutics (Welwyn Garden City, UK), specializes in discovering new medicines that targeting previously undruggable or challenging GPCRs. In addition to discovering small-molecule drugs, Heptares, working with monoclonal antibody (MAb) leaders such as MorphoSys and MedImmune, is working to discover MAbs that act as modulators of GPCRs. Among Heptares’ targets are several GPCRs with peptide ligands.

Meanwhile, Kyowa Hakko Kirin Co., Ltd. has developed the MAb drug mogamulizumab (trade name Poteligeo), which is approved in Japan for treatment of relapsed or refractory adult T-cell leukemia/lymphoma. Mogamulizumab targets CC chemokine receptor 4 (CCR4).

Thus, aided in part by structural biology, the discovery of novel drugs that target GPCRs–including those with protein or peptide targets such as chemokine receptors–continues to make progress.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company,  please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Cancer immunotherapy: the star of the ASCO Annual Meeting two years in a row!

PD-L1

PD-L1

On June 28, 2012 we published an article on this blog entitled “Cancer Immunotherapy: The Star Of The 2012 ASCO Annual Meeting”. Now comes the American Society of Clinical Oncology (ASCO) 2013 Annual Meeting, which took place from May 30 to June 3, 2013.

As in 2012, cancer immunotherapy was the star of the meeting.

In our June 2012 article, we focused on experimental monoclonal antibody (MAb) drugs that target the cell surface receptors programmed cell death-1 (PD-1) and programmed cell death-1 ligand (PD-L1). PD-1 is a member of the CD28/CTLA4 family of T cell regulators. Like CTLA4, the target of ipilimumab, PD-1 is a negative regulator of T-cell receptor signals. When PD-L1, which is a protein on the surface of some tumor cells, binds to PD-1 on T cells that recognize antigens on these tumor cells, this results in the blockage of the ability of the T cells to carry out an anti-tumor immune response. Anti-PD-1 MAb binds to PD-1 on T cells, thus preventing PD-L1 on tumor cells from binding to the PD-1 and initiating an inhibitory signal. Anti-tumor T cells are then free to initiate immune responses against the tumor cells. This mechanism of action is completely analogous to that of ipilimumab, which binds to CTLA4 and thus prevents negative signaling from that molecule.

Anti-PD-L1 therapeutics bind to PD-L1 on tumor cells. Ira Mellman (vice-president of research oncology at Genentech), believes that anti-PD-L1 might have fewer adverse effects than anti-PD-1. That is because anti-PD-L1 would target tumor cells while leaving T cells free to participate in immune networks that work to prevent autoimmune reactions.

Three experimental drugs in this area of immunotherapy were a main focus at ASCO in 2013. They are:

  • BMS’ anti-PD-1 agent nivolumab (BMS-936558, MDX-1106), which we had discussed in our 2012 ASCO article.
  • Merck’s anti-PD-1 agent lambrolizumab (MK-3475)
  • Roche/Genentech’s anti-PD-L1 agent MPDL3280A

We shall focus on these three agents in this article.

Competition between BMS’ nivolumab and Merck’s lambrolizumab

As highlighted in the 2013 ASCO meeting and in reports by industry commentators such as FierceBiotech, there is a keen race between BMS and Merck to be the first to market an anti-PD-1 agent.

At the ASCO 2013 meeting, BMS researchers and their colleagues reported that a third of the patients in a Phase 1 trial of nivolumab saw tumors shrink at least 30%. They also reported that patients with solid tumors [metastatic melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC)] showed high rates of 2 year overall survival–44% for melanoma, 32% for NSCLC, and 52% for RCC (clinical trial NCT00730639).

In a first Phase 1 study of a combination therapy of nivolumab with ipilimumab in metastatic melanoma, BMS researchers and their colleagues reported that the two agents could be administered in combination safely. Clinical activity for the combination therapy appeared to exceed that of published monotherapy data for each of the two agents, with greater or equal to 80% tumor reduction at 12 weeks in 30% (11/37) of patients. In addition to the ASCO 2013 presentation, the results of this combination therapy trial were published online in the New England Journal of Medicine.

According to Fierce Biotech, BMS has 6 late-stage studies under way for nivolumab, with fast-track status in place for melanoma, lung cancer and kidney cancer.

Meanwhile, Merck announced in a June 2, 2013 press release the presentation at ASCO 2013 of interim data from a Phase 1B study evaluating its anti-PD-1 agent lambrolizumab in patients with advanced melanoma. The data was presented by Antoni Ribas, M.D., Ph.D. (Jonsson Comprehensive Cancer Center, University of California, Los Angeles). in addition to the ASCO 2013 presentation, this study was published online in the New England Journal of Medicine.

A total of 135 patients with advanced melanoma were treated. Most of the adverse events seen in the study were low grade. The confirmed response rate across all dose cohorts was 38%. The highest confirmed response rate (52%) was seen in the cohort that received the highest dose (10 mg per kilogram every 2 weeks). Ten percent of the patients in the highest-dose group achieved a complete response, with response duration ranging from 28 days to 8 months.

Response rates did not differ significantly between patients who had received prior ipilimumab treatment and those who had not. Responses were durable in the majority of patients; 81% of the patients who had a response (42 out of of 52 total) were still receiving treatment at the time of analysis in March 2013. The overall median progression-free survival among the 135 patients was over 7 months.

According to Fierce Biotech, Merck now has four clinical studies under way for lambrolizumab, including a  Phase 2 trial in melanoma and Phase 1 trials in ipilimumab-naïve patients with triple-negative breast cancer, metastatic bladder cancer and head and neck cancer. The company, which has won breakthrough drug designation from the FDA for lambrolizumab, believes that the ongoing 500-patient Phase 2 melanoma study could provide enough positive data to win FDA approval. Merck is also preparing applications for late-stage clinical trials in melanoma and non-small cell lung cancer, which are planned to launch in the third quarter of 2013.

Roche/Genentech’s anti-PD-L1 agent MPDL3280A

Genentech researchers and their collaborators presented data on a clinical study of MPDL3280A in patients with metastatic melanoma at ASCO 2013. In addition to the ASCO 2013 presentation and abstract, The Angeles Clinic and Research Institute (Los Angeles, CA) published a press release about the study. Omid Hamid, M.D. of The Angeles Clinic and Research Institute made the oral presentation at the ASCO meeting.

This study was a Phase 1, multicenter, first in human, open-label, dose escalation study (clinical trial NCT01375842), which is still ongoing. It was primarily designed to assess  safety, tolerability, and pharmacokinetics of MPDL3280A in patients with metastatic melanoma. The drug was found to be well tolerated. 35 patients who began treatment at doses of 1-20 mg/kg and were enrolled prior to Jul 1, 2012 were evaluable for efficacy. An overall response rate of 26% (9/35) was observed, with all responses ongoing or improving. Some responding patients experienced tumor shrinkage within days of initial treatment. The 24-week progression-free survival was 35%. Several other patients had delayed antitumor activity after apparent tumor progression. Of three initial patients treated with a combination of MPDL3280A and vemurafenib (Daiichi Sankyo/Genentech’s Zeboraf, a targeted kinase inhibitor), two experienced tumor shrinkage, including 1 complete response. The researchers concluded that further assessment of MPDL3280A as monotherapy and combination therapy is warranted. A Phase 1 study (NCT01656642) of a combination therapy of MPDL3280A and vemurafenib in patients with previously untreated BRAFV600-mutation positive metastatic melanoma is ongoing.

Data was also presented at ASCO 2013 on the efficacy of MPDL3280A in other solid tumors. According to Roy S. Herbst, M.D. Ph.D., (Yale Cancer Center and Smilow Cancer Hospital at Yale-New Haven) MPDL3280A showed significant anti-tumor activity and was well tolerated in patients with such cancers as NSCLC, melanoma, colorectal cancer, gastric cancer, and RCC. 29 of 140 evaluable patients (21%) exhibited tumor shrinkage, with the highest overall responses in patients with NSCLC and melanoma. Of the 29 responders, 26 patients continued responding as of their last assessment.

Researchers have also been studying PD-L1 expression levels as a potential biomarker to identify likely responders. As outlined by Dr. Herbst, responses appeared to be better among patients with higher levels of PD-L1 expression. The response rate among PD-L1-positive patients was 36% (13 of 36 patients), compared with 13% (9 of 67 patients) who were PD-L1-negative. The role that PD-L1 expression might play as a biomarker is still being explored, including attempting to determine the best way to measure the protein and other related criteria.

In addition to the Phase 1 trial of MPDL3280A/vemurafenib combination therapy in melanoma, Genentech is sponsoring a Phase 1 trial of MPDL3280A in combination with bevacizumab (Genentech/Roche’s Avastin, an angiogenesis inhibitor that targets vascular endothelial growth factor) or with bevacizumab plus chemotherapy (clinical trial NCT01633970). Genentech is also sponsoring a Phase 2 clinical trial (NCT01846416) of MPDL3280A in patients With PD-L1-positive advanced NSCLC.

Conclusions

The field of immunotherapeutic MAbs for cancer, which target negative regulators of T-cell receptor signals, continues to advance. The approval and marketing of ipilimumab provides an important proof-of-principle for this approach. Now the field is advancing to include agents that target PD-1 and its negative regulator PD-L1. Studies of BMS’ PD-1 inhibitor nivolumab have advanced as far as Phase 3, and of Merck’s lambrolizumab as far as Phase 2. Meanwhile, Roche/Genentech’s PD-L1 inhibitor MPDL3280A has reached Phase 2.

However, the in terms of clinical trial data, it is still too early to meaningfully determine the efficacy of any of the PD-1 and PD-L1 inhibitor drugs. The meaningful data will come from randomized Phase 3 trials, based on overall survival rather than tumor response rate as in currently reported trials (with the exception of the Phase 1 results of clinical trial NCT00730639 of nivolumab described earlier, which included measures of overall survival).

Nevertheless, this is an extremely exciting field, and researchers, companies, and patient communities have high expectations of success.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company,  please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

FDA proposes accelerated approval of early-stage Alzheimer’s drugs based on cognitive improvement alone

Pittsburgh compound B staining in AD. Source: National Institute on Aging/NIH.

Pittsburgh compound B staining in AD. Source: National Institute on Aging/NIH.

In our February 28, 2013 article on the Biopharmconsortium Blog, we discussed the FDA’s February 7, 2013 Draft Guidance for Industry entitled “Alzheimer’s Disease: Developing Drugs for the Treatment of Early Stage Disease”.

This document had been distributed for comment purposes only, and the FDA has been seeking public comment on the draft guidance for 60 days following publication.

As we discussed, by issuing this Draft Guidance, the FDA added its voice to that of an ever-increasing segment of the scientific community that calls for a new focus on conducting clinical trials in early-stage Alzheimer’s disease (AD). This is in order to  focus industry R&D on developing treatments for patients whose disease is in a stage prior to the development of extensive irreversible brain damage. It is in this early stage of disease in which researchers believe that new drugs have the best chance of providing benefits to patients, by preventing further damage to the brain.

In our February 28, 2013 article, we also discussed several clinical trials being carried out by industry and academic researchers in early-stage AD. These trials should allow the scientific and medical community to answer the question as to whether treating patients with pre-AD or very early-stage AD with anti-amyloid MAb drugs can have a positive effect on the course of the disease, and slow or prevent cognitive decline.

Readers of our article may have noticed that the February 7, 2013 Draft Guidance was somewhat vague or confused. That is because there is currently no evidence-based consensus as to which biomarkers might be appropriate to support clinical findings in trials in early AD. Moreover, in “pre-AD” or very early-stage AD (i.e., before the onset of overt dementia) disease-related impairments are extremely challenging to assess accurately. Thus both measuring clinical outcomes and assessment via biomarkers in very early-stage AD are fraught with difficulty, making determination of drug efficacy very difficult.

In issuing the Draft Guidance, The FDA appeared to be seeking guidance from industry and from the academic community on how these issues might be resolved. As we said in our article, the early-stage AD trials now in progress might help the scientific and medical community, and the FDA, with issues of evaluation of biomarkers and clinical outcome measures in determining disease prognosis and the efficacy of drug treatments.

More recently–on March 13, 2013–the FDA proposed a further modification of its proposed guidelines for regulation of early-stage AD therapeutics. This was published online in an article in the New England Journal of Medicine (NEJM), entitled “Regulatory Innovation and Drug Development for Early-Stage Alzheimer’s Disease”, by Nicholas Kozauer, M.D. and Russell Katz, M.D. (As we stated in our earlier article, Dr.Katz is the director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research. Dr. Kozauer is a Clinical Team Lead in the same division of the FDA.)

The new proposal attempts to deal with some of the apparent confusion in the February 7, 2013 Draft Guidance, and to facilitate the development and approval of new drugs for early-stage AD. The NEJM article notes that traditional measures of AD drug efficacy at the FDA had included assessment both of improved cognition and improvements in function. Specifically, as stated by a New York Times article discussing the new FDA proposal, “cognition” refers to such mental processes as memory and reasoning (as assessed by various tests), and “function” refers to performing such day-to-day activities as cooking, dressing or bathing.

In the FDA’s March 13, 2013 NEJM article, the authors note that researchers and regulatory agencies “simply do not yet have drug-development tools that are validated to provide measures of function in patients with Alzheimer’s disease before the onset of overt dementia”. Thus, although one can test early-stage AD patients for improvements in cognition with the appropriate tests, testing for deficits and improvements in function is extremely difficult.

The authors of the NEJM article therefore suggest that it might be feasible that a drug for treating early-stage AD be approved via the FDA’s accelerated approval pathway, on the basis of assessment of cognitive outcome alone. The agency’s accelerated-approval pathway allows drugs that address an unmet medical need to be approved on the basis of a surrogate or an intermediate clinical endpoint–in this case, a sensitive measure of improvement in cognition. Drugs approved via “accelerated approval” must be subjected to postmarketing studies to verify the clinical benefit. This regulatory pathway might facilitate the approval of treatments that appear to be effective in early AD, when patients might be expected to derive a greatest benefit than after the development of overt dementia.

With respect to selection of patients for trials in early-stage AD, the authors of the NEJM article suggest that (based on “the consensus emerging within the AD research community”) clinical diagnosis of early cognitive impairment be combined with appropriate biomarkers. These biomarkers might include brain amyloid load [as measured by positron-emission tomography (PET)] and cerebrospinal fluid levels of β-amyloid and tau proteins. The FDA places a high priority on efforts by the researchers to qualify such biomarkers in clinical trial design in early-stage AD.

The author of the New York Times article, veteran science and medicine reporter Gina Kolata, says that the FDA’s new proposal could “help millions of people at risk of developing [AD] by speeding the development and approval of drugs that might slow or prevent it.”

She also says that the proposal could be a boon for the pharmaceutical industry and AD researchers. They have often been hampered by regulations that left them uncertain of how to get drugs tested and approved for early-stage AD. Not only might anti-AD therapies provide greater benefit to patients with early-stage AD than with later stage disease, but clinical trials in early-stage AD would have a greater potential for success–provided that researchers had appropriate means of determining efficacy in early-stage AD. The new FDA proposal may increase the likelihood of identifying such appropriate means.

As pointed out in the Times article, several leading AD researchers agree, with some important caveats. For example, AD researcher P. Murali Doraiswamy, M.D. (Duke University School of Medicine) said that the new proposed regulations would lead to more clinical trials, and more motivation now to invest in the AD field. However, many companies never manage to do postmarking studies required for drugs given accelerated approval, and such studies might not be randomized clinical trials as required in gaining approval of the drugs in the first place.

Sean Bohen, M.D., Ph.D. (Senior Vice President for Early Development at Genentech) was very positive about the proposed new FDA policy, but wondered how researchers could develop appropriate tests to identify subtle cognitive changes in early AD or pre-AD. Nevertheless, he said, “We have to start somewhere.”

Thus clinical trials in early-stage AD, and development of regulatory frameworks for approval and postmarketing studies of agents that emerge from these trials, remain a work in progress.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or an initial one-to-one consultation on an issue that is key to your company’s success, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

FDA publishes Draft Guidance on developing drugs for early stages of Alzheimer’s disease

 

Normal and Alzheimer's brains compared.

Normal and Alzheimer’s brains compared.

Once again, approaches to improving clinical trials for candidate disease-modifying drugs for Alzheimer’s disease (AD) are in the news. On February 7, 2013, the FDA issued a Draft Guidance for Industry entitled “Alzheimer’s Disease: Developing Drugs for the Treatment of Early Stage Disease”.

This document has been distributed for comment purposes only, and the FDA is seeking public comment on the draft guidance for 60 days.

The wording of the Draft Guidance illustrates the extreme difficulty of defining populations with pre-AD or very early-stage AD, and of demonstrating the efficacy of a drug in ameliorating early-stage disease, and/or in preventing its progression to later-stage disease. The document states that the FDA is “open to considering the argument that a positive biomarker result (generally included as a secondary outcome measure in a trial) in combination with a positive finding on a primary clinical outcome measure may support a claim of disease modification in AD.”

However,  there is currently no evidence-based consensus as to which biomarkers might be appropriate to support clinical findings in trials in early AD. Moreover, in “pre-AD” or very early-stage AD (i.e., before the onset of overt dementia) mild disease-related impairments are extremely challenging to assess accurately. Thus both measuring clinical outcomes and assessment via biomarkers in very early-stage AD are fraught with difficulty, making determination of drug efficacy extremely difficult. The FDA thus appears to be seeking guidance from industry and from the academic community on how these knotty problems might be solved.

The move toward conducting clinical trials in early-stage AD patients

By issuing the Draft Guidance, the FDA adds its voice to that of an ever-increasing segment of the scientific community that calls for a new focus on conducting clinical trials in early-stage AD. We discussed this trend in our August 19, 2012 and August 28, 2012 articles on the Biopharmconsortium Blog.

As we discussed, this trend is driven in part by the Phase 3 failures of Pfizer/Janssen’s bapineuzumab and Lilly’s solanezumab in 2012. Now–in February 2013–Russell Katz, M.D. (director of the Division of Neurology Products in the FDA’s Center for Drug Evaluation and Research) says, “The scientific community and the FDA believe that it is critical to identify and study patients with very early Alzheimer’s disease before there is too much irreversible injury to the brain. It is in this population that most researchers believe that new drugs have the best chance of providing meaningful benefit to patients.”  In line with this statement, the FDA refused to entertain Lilly’s  secondary analysis of early stage patients in the solanezumab study that we discussed in our August 28, 2012 blog article. Instead, the FDA mandated that Lilly conduct a new Phase 3 trial that will exclude the moderate-stage patients who hadn’t responded, and focus only on early-stage patients.

Recent news on clinical trials in early-stage AD

Despite the difficulties highlighted in the Draft Guidance in conducting clinical trials in early-stage AD patients, three research groups are actually conducting such trials. We outlined these studies in our August 28, 2012 blog article, and discussed one of these studies, the one begin carried out by Genentech, in greater detail in our August 19 2012 article.

The three studies are:

  • Roche/Genentech’s Phase 2a trial of its its anti-amyloid MAb crenezumab, in presymptomatic members of a large Colombian kindred who harbor a mutation in presenilin 1 (PS1) that causes dominant early−onset familial AD.
  • Studies conducted in conjunction with the Dominantly Inherited Alzheimer Network (DIAN), a consortium led by researchers at Washington University School of Medicine (St. Louis, MO). This study will include people with mutations in any of the three genes linked to early-stage, dominantly-inherited AD–PS1, PS2, and amyloid precursor protein (APP). Initial studies focused on changes in biomarkers and in cognitive ability as a function of expected age of AD onset in people with these mutations. These included changes in concentrations of amyloid-β1–42 (Aβ42) in cerebrospinal fluid (CSF), and amyloid accumulation in the brain. In the first stage of the actual trial, three drugs (which have not yet been selected) will be tested in this population, and changes in biomarkers and cognitive performance will be followed.
  • The Anti-Amyloid Treatment of Asymptomatic Alzheimer’s (A4) trial, will involve treating adults without mutations in any of the above three genes, whose brain scans show signs of amyloid accumulation. A4 is thus designed to study prevention of sporadic AD (by far the most common form of the disease). It will enroll 500 people age 70 or older who test positive on a scan of amyloid accumulation in the brain. (This is in contrast to the two trials in subjects with gene mutations, who are typically in their 30s or 40s.) A4 will also have a control arm of 500 amyloid-negative subjects. Amyloid-positive and control subjects will be entered into a three-year double-blind clinical trial that will look at changes in cognition with drug treatment. The A4 researchers [led by Reisa Sperling, Brigham and Women’s Hospital/Harvard University (Boston, MA), and Paul Aisen, University of California, San Diego] planned to select a drug for testing by December 2012.

Now there is more recent news on two of these trials.

1. On December 13, 2012, the Los Angeles Times reported that Genentech and its collaborators [affiliated with the University of Antioquia medical school (Medellin, Colombia), the University of California at Los Angeles (UCLA), and the Banner Alzheimer’s Institute (Phoenix, AZ)] will begin their $100 million clinical trial of crenezumab with 100 Colombians who carry the PS1 mutation in the spring of 2013. Genentech is contributing $65 million of the study’s $100-million cost. The NIH and the Banner Alzheimer’s Institute (Phoenix, AZ) are financing the remainder.

This story was also reported on December 14, 2012 by Fierce Biotech.

The design of the trial calls for 100 additional patients in Colombia with the same Alzheimer’s-related gene to receive a placebo, and an equal number of other at-risk patients without the gene to take crenezumab.  A branch of the trial will include U.S. patients as well. A “branch study” will also be conducted at UCLA, where researchers have discovered a similar genetic disposition among members of an extended family from Jalisco, Mexico. Some 30 individuals from this family who have immigrated to Southern California could participate. Around 150 other U.S. patients with similar mutations will also participate in the trial.

The trial is designed to provide evidence that targeting amyloid with crenezumab at an early stage or even before patients show signs of dementia can have a positive effect on the course of disease.

2. On January 18, 2013, Fierce Biotech reported that the researchers conducting the A4 study have chosen Lilly’s solanezumab as as the first therapeutic drug candidate to be evaluated in the trial. The A4 trial’s principal investigator, Reisa Sperling said that the researchers chose solanezumab (after considering a number of anti-amyloid drugs) because the compound has a good safety profile, and appeared to show a modest clinical benefit in the mild AD patients in Lilly’s Phase 3 trial. The A4 researchers’ confidence in solanezumab grew when this was confirmed via an independent academic analysis by the Alzheimer’s Disease Cooperative Study (ADCS), a consortium of academic Alzheimer’s disease clinical trial centers. The ADCS, which was established by NIH, will help facilitate the A4 trial.

The A4 researchers hope that starting treatment with solanezumab before symptoms are present, as well as treating for a longer period of time, will slow cognitive decline and ultimately prevent AD dementia.

After the failure of solanezumab in Lilly’s own Phase 3 studies, and the FDA’s rebuff of the company’s secondary analysis of early stage patients, the A4 study’s choice of solanezumab gives the drug a new lease on life. Meanwhile, Lilly will be continuing its own clinical trial program for solanezumab.

Conclusions

The three clinical trials discussed in this article should allow the scientific and medical community to answer the question as to whether treating patients with pre-AD or very early-stage AD with anti-amyloid MAb drugs can have a positive effect on the course of the disease, and slow or prevent cognitive decline. The studies may also help the scientific and medical community, and the FDA, with issues of evaluation of biomarkers and clinical outcome measures in determining disease prognosis and the efficacy of drug treatments. Given the large size and rapid growth of the at-risk population, finding safe and efficacious disease-modifying preventives and treatments for AD is of increasing urgency.

________________________________

As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or an initial one-to-one consultation on an issue that is key to your company’s success, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Here we go again–Lilly’s Alzheimer’s drug solanezumab fails to show efficacy in Phase 3, but company is “encouraged” by secondary analysis

 

Amyloid precursor protein (APP)

As we mentioned in our August 19, 2012 article on Alzheimer’s disease (AD), the results of Phase 3 trials of Lilly’s amyloid-targeting monoclonal antibody (MAb) drug solanezumab, had been expected soon.

On August 24 2012, Lilly announced the top-line results of the two Phase 3, double-blind, placebo-controlled EXPEDITION trials of solanezumab in patients with mild-to-moderate Alzheimer’s disease. The primary endpoints, both cognitive and functional, were not met in either of these trials.

However, a pre-specified secondary analysis of pooled data across both trials showed statistically significant slowing of cognitive decline in the overall study population, and pre-specified secondary subgroup analyses of pooled data across both studies showed a statistically significant slowing of cognitive decline in patients with mild Alzheimer’s disease, but not in patients with moderate Alzheimer’s disease.

These results were reported in a press release.  What was absent was data from the trials. However, the Alzheimer’s Disease Cooperative Study (ADCS), (an academic national research consortium) will present its independent analysis of the data from the EXPEDITION studies at the American Neurological Association (ANA) meeting in Boston on October 8, 2012, and at the Clinical Trials on Alzheimer’s Disease (CTAD) meeting in Monte Carlo, Monaco, on October 30, 2012.

Once again, an amyloid pathway-targeting drug for Alzheimer’s disease that was taken into Phase 3 trials despite Phase 2 results that showed no statistically significant efficacy has failed in Phase 3. Solanezumab joins a list of such failed drugs that includes Myriad Pharmaceuticals’ Flurizan (tarenflurbil), Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid), and as of July 2012, Pfizer/Janssen’s bapineuzumab (“bapi”). Nevertheless, as in the Phase 2 results with bapi, Lilly sees hope for the drug in the results of secondary analyses.

On the day of the Lilly announcement, August 24 2012, Lilly executives and stock analysts turned the results of these trials into something “positive”, as the result of the secondary analysis. This resulted in a one-day 3.4 percent increase in the price of Lilly stock. However, the results of the secondary analysis do not give Lilly any basis for going to the FDA with a New Drug Application (NDA) for solanezumab. Nor do they provide any realistic hope for AD patients, the physicians who treat them, or caregivers of AD patients.

At best, Lilly’s secondary analysis gives rise to a hypothesis–that solanezumab–and presumably other anti-amyloid MAbs–will be effective in treating earlier-stage AD patients, especially those who have not suffered extensive, irreversible brain damage. This is the very same hypothesis that is now being tested by Roche/Genentech in its clinical trials of its anti-amyloid MAb crenezumab, as we discussed in our August 19, 2012 article. Genentech is testing its drug candidate in a Phase 2a trial in a very special population–members of a large Colombian kindred who harbor a mutation in presenilin 1 (PS1) that causes dominant early−onset familial AD.

A News Focus article in the 17 August 2012 issue of Science, written by science writer Greg Miller, PhD, discusses three upcoming clinical trials designed to test the “treat early-stage or presymptomatic AD with anti-amyloid MAbs” hypothesis. One of these studies is the Genentech trial of crenezumab in the extended family in Colombia.

Another of these studies is being conducted in conjunction with the Dominantly Inherited Alzheimer Network (DIAN), a consortium led by researchers at Washington University School of Medicine (St. Louis, MO). This study will include people with mutations in any of the three genes linked to early-stage, dominantly-inherited AD–PS1, PS2, and amyloid precursor protein (APP).

Initial studies, published ahead of print in the July 11 issue of the New England Journal of Medicine (NEJM) looked at changes in biomarkers and in cognitive ability as a function of expected age of AD onset in people with these mutations. Concentrations of amyloid-β1–42 (Aβ42) in the cerebrospinal fluid (CSF) appeared to decline 25 years before expected symptom onset. This decrease may reflect impaired clearance of Aβ42 from the brain, which may be a factor in the amyloid plaque increase that is associated with AD. Amyloid accumulation in the brain was detected 15 years before expected symptom onset. Other biomarkers, as well as cognitive impairment, were also followed in the study published in the NEJM. In the first stage of the actual trial, three drugs (which have not yet been selected) will be tested in this population, and changes in biomarkers and cognitive performance will be followed.

The third study, known as the Anti-Amyloid Treatment of Asymptomatic Alzheimer’s (A4) trial, will involve treating adults without mutations in any of the above three genes, whose brain scans show signs of amyloid accumulation. A4 is thus designed to study prevention of sporadic AD (by far the most common form of the disease). It will enroll 500 people age 70 or older who test positive on a scan of amyloid accumulation in the brain. (This is in contrast to the two trials in subjects with gene mutations, who are typically in their 30s or 40s.) A4 will also have a control arm of 500 amyloid-negative subjects. Amyloid-positive and control subjects will be entered into a three-year double-blind clinical trial that will look at changes in cognition with drug treatment. The A4 researchers [led by  Reisa Sperling, Brigham and Women’s Hospital/Harvard University (Boston, MA), and Paul Aisen, University of California, San Diego] plan to select a drug for testing by December 2012.

If Lilly wishes to test solanezumab in early-stage (or presymptomatic) sporadic AD, it will need to follow a similar methodology to the studies outlined in the new Science article, especially with respect to the use of biomarkers to define “early-stage” AD and to track the effects of the drug. Studies such as the DIAN biomarker study published in the NEJM used the positron emission tomography (PET) ligand Pittsburgh Compound-B (PiB-C11), to image amyloid plaques. However, the use of this compound is limited by the short half-life of carbon-11 (20.4 minutes). A new PET amyloid imaging agent, Amyvid (florbetapir F18 Injection) was developed by Lilly and approved by the FDA in April 2012. This compound contains fluorine-18, which has a half-life of 109.8 minutes. A recent study indicates that Amyvid provides comparable information to PiB-C11. If Lilly wishes to conduct new studies of solanezumab in early-stage or presymptomatic sporadic AD, it may wish to use Amyvid, as suggested in a comment to an August 24, 2012 solanezumab post in Derek Lowe’s blog “In the Pipeline”. However, the FDA, in its press release announcing the approval of Amyvid, warns that increased amyloid plaque content (as detected by Amyvid or Pittsburgh Compound-B) may be present in the brains of patients with non-AD neurologic conditions, and in older people with normal cognition. Thus defining or detecting “early-stage (or presymptomatic) sporadic AD” is difficult.

In any case, for Lilly to follow up on its secondary analyses of the Phase 3 clinical trials of solanezumab will necessitate additional long and expensive clinical trials, with no assurance of success. Lilly executives will need to determine if such a course is worth the risk, or whether it should invest in other R&D efforts that might have a higher probability of success.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

New genetics study supports the amyloid hypothesis of Alzheimer’s disease–but the drugs still don’t work!

 

The APP processing pathway

An exciting new study on Alzheimer’s disease (AD) was published in the 2 August issue of Nature. The study was carried out by researchers at deCode Genetics (Reykjavik Iceland) and their collaborators at Genentech and several academic institutions. A News and Views article by leading AD researcher Bart De Strooper and genomics researcher Thierry Voet (both at KU Leuven, Leuven, Belgium) analyzes this study and its implications.

Amyloid plaques are a central feature of AD.  They largely consist of amyloid-β (Aβ) peptides. Aβ peptides are formed via sequential proteolytic processing of the amyloid precursor protein (APP), catalyzed by two aspartyl protease enzymes–β-secretase and γ-secretase.  The β-site APP cleaving enzyme 1 (BACE1) cleaves APP predominantly at a unique site. However, γ-secretase cleaves the resulting carboxy-terminal fragment at several sites, with preference for positions 40 and 42. This leads to formation of amyloid-β1–40 (Aβ40) and Aβ1–42 (Aβ42) peptides. APP processing to yield Aβ peptides is illustrated by the figure at the top of this article.

By studying rare, familial cases of early-onset AD, human geneticists have identified three disease genes in these conditions— genes for APP, and for two presenilins, PS1 and PS2. The presenilins are components of γ-secretase, which exists as an intramembrane protease complex. Mainly because of these genetic studies, as well as studies in animal models and postmortem studies of AD brains, the majority of AD researchers have focused on the APP processing pathway and/or on aggregation of Aβ to form plaques as intervention points for therapeutic strategies. The hypothesis that this is the central AD disease pathway is called the “amyloid hypothesis”.

Up until the publication of the new deCode report, of the 30-odd coding mutations in APP that have been found, around 25 are pathogenic, usually resulting in autosomal dominant early-onset Alzheimer’s disease. Coding mutations at or near the β- or γ-proteolytic sites have appeared to result in overproduction of either total Aβ or a shift in the Aβ40:Aβ42 ratio towards formation of Aβ42, which is the more toxic of the two Aβ peptide. Until now, mutations in APP have not been implicated in the common, late-onset form of Alzheimer’s disease.

In the new deCode study, the researchers studied coding variants in APP in a set of whole-genome sequence data from 1,795 Icelanders. They identified a single nucleotide polymorphism (SNP), designated as rs63750847. The A allele of this SNP (rs63750847-A) results in an alanine to threonine substitution at position 673 in APP (A673T). The A673T mutation was found to be significantly more common in the elderly (age 85-100) control group (i.e., those without AD) than in the AD group. The researchers therefore concluded that the mutation is protective against AD.

The researchers also found that in a cohort of individuals over 80, those who were heterozygous for the A673T mutation performed better in a test of mental capacity than did control subjects. The authors concluded that the A673T mutation not only protects against AD, but also against the mild cognitive decline that is normally associated with old age.

In cellular studies (i.e., studies in cultured cells transfected with genes coding for wild type or mutant APP) and in biochemical studies, the researchers found that APP carrying the A673T mutation undergoes about 40% less cleavage by BACE1 than does wild-type APP, resulting in 40% less production of both Aβ40 and Aβ42.

The researchers conclude that the strong protective effect of the A673T mutation against AD provides proof of principle for the hypothesis that reducing the β-cleavage of APP (e.g., by use of BACE1 inhibitors, such as those being  developed by some pharmaceutical companies) may protect against the disease. (However, success in developing BACE1 inhibitors has been elusive.) Moreover, since the A673T allele also protects against cognitive decline in elderly individuals who do not have AD, AD and age-related mild cognitive decline may be mediated through the same or similar mechanisms.

Despite this compelling genetic finding, amyloid pathway-targeting drugs have not shown efficacy in Phase 3 trials

In our January 26, 2010 blog article, we discussed Phase 2 clinical trials of bapineuzumab, a monoclonal antibody (MAb) drug that is specific for Aβ, in mild to moderate AD. In that article, we referred to the drug as “Elan/Wyeth’s bapineuzumab”, after the original developers of the drug. As the result of mergers and acquisitions, the drug is now referred to as “Pfizer/Janssen’s bapineuzumab”. Many commentators call it “bapi” for short.

As we discussed in that article, the overall result of the Phase 2 trial was that there was no difference in cognitive function between patients in the bapi-treated and the placebo groups. However, the study did not have sufficient statistical power to exclude the possibility that there was such a difference. Retrospective analysis of the data from the trial suggested that bapi-treated patients who were not carriers of the apolipoprotein E epsilon4 allele (ApoE4) showed improved cognitive function as compared to placebo treatment. Given that this conclusion was reached via retrospective analysis, the idea that the bapi was efficacious in ApoE4 noncarriers was only a hypothesis, which would require prospective clinical trials to confirm. Janssen and Pfizer had been conducted large Phase 3 trials of bapi, which they prospectively segregated into ApoE4 carrier and noncarrier groups in order to test this hypothesis.

As of the past several weeks, the results of these Phase 3 trials have come in. On July 23rd, 2012, Pfizer announced the top-line results of an 18-month Janssen-led Phase 3 study of intravenous bapi in approximately 1,100 patients with mild to moderate Alzheimer’s disease who carry at least one ApoE4 allele. The drug failed to meet its co-primary endpoints (change in cognitive and functional performance compared to placebo) in that study. On August 6, 2012, Pfizer announced the top-line results of the corresponding Phase 3 study of intravenous bapi in patients with mild-to-moderate Alzheimer’s disease who do not carry the ApoE4 genotype. Once again, the co-primary clinical endpoints were not met. Based on these results, the companies decided to discontinue all other intravenous bapi studies in patients with mild-to-moderate Alzheimer’s disease.

The bapi development program continues a history of amyloid pathway-targeting drugs that were taken into Phase 3 trials despite Phase 2 results that showed no statistically significant efficacy. For example, we cited the cases of Myriad Pharmaceuticals’ Flurizan (tarenflurbil) and Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid) in our January 26, 2010 blog article.

Leading industry commentator Matthew Herper of Forbes referred to the failure of bapi as “the latest piece of evidence of the drug industry’s strange gambling problem.” Johnson & Johnson (the parent company of Janssen) spent more than $1 billion to invest in Elan and get one-quarter of bapi, and Wyeth (later Pfizer) and Elan put the drug into Phase 3, despite the Phase 2 failure of bapi.

The temptation for pharmaceutical companies to take a chance on an AD drug such as bapi, Flurizan, and Alzhemed is driven by the complete lack of disease-modifying AD drugs, and the thinking that even a not-very-effective drug that receives FDA approval might generate billions of dollars in annual sales. In the case of bapi there was also that tantalizing suggestion that bapi might show efficacy in the subset of patients who lacked ApoE4.

In an August 16, 2012 article in Forbes, Dr. John LaMattina (the former President of Pfizer Global R&D) engages in informed speculation as to why bapi was moved into Phase 3. Dr. LaMattina (in contrast to critics like Mr. Herper, who discounted the ApoE4 retrospective analysis as “data-dredging” that was “likely to be due to chance”) referred to the efficacy signal of the Phase 2 trials as “mixed” due to the ApoE4 analysis. He stated that such “mixed results” present an “agonizing” dilemma for a pharmaceutical company.

In deciding whether to go forward Phase 3 trials of bapi, Dr. LaMattina further speculates that the decision might have been influenced by stakeholders such as AD patient advocates, and scientists who strongly believed in the science behind bapi, especially the amyloid hypothesis. Moreover, bapi had been shown to be relatively safe. In addition, dropping bapi would have caused public relations damage. Dr. LaMattina concludes, based on this analysis, “…this was a situation where these companies were in possession of a relatively safe drug, with a modest chance of success in being efficacious in what may be the biggest scourge that society will face.  How can you not make this investment?” He reminds us that pharmaceutical R&D “is a high risk, high reward business”.

Nevertheless, bapi joined Flurizan and Alzhemed on the list of high-profile amyloid-pathway failures. Now a Phase 3 trial of Lilly’s solanezumab, another MAb drug that targets Aβ, is nearing completion, with the results expected in September. Published Phase 2 results were designed to test safety, not efficacy, and 12 weeks of drug treatment gave no change in cognitive function. Although the results of the Phase 3 trial will not be known until they are reported, analysts expect the drug to fail because of its similarity to bapi.

Why don’t amyloid pathway-targeting drugs show efficacy in clinical trials, despite the compelling genetic evidence for the amyloid hypothesis?

The almost standard answer to that question given by scientists and clinicians who support the amyloid hypothesis is that we have been testing the drugs too late in the course of AD progression, after the damage to the brain has become irreversible. Roche/Genentech is testing this idea in its clinical trials of its drug candidate crenezumab (licensed from AC Immune), which is yet another MAb drug that targets Aβ. In a 5-year Phase 2a clinical trial, Genentech is testing intravenous crenezumab in 300 cognitively healthy individuals from a large Colombian kindred who harbor the Glu280Ala (codon 280 Glu to Ala substitution) PS1 mutation. This mutation causes dominant early−onset familial AD, and is associated with increased levels of Aβ42 in plasma, skin fibroblasts, and the brain. Family members with this mutation begin showing cognitive impairment around age 45, and full dementia around age 51.

Genentech is conducting this trial in collaboration with the Banner Alzheimer’s Institute and the National Institutes of Health. The company says that this trial is the first-ever AD prevention study in cognitively healthy individuals. Genentech further says that the trial may help to determine if the amyloid hypothesis is correct–more specifically, it may help to determine if a drug that works by depleting amyloid plaques can be effective in preventing and/or treating AD.

Moreover, Genentech states that there is significant unmet medical need within this Colombian population. This large extended family may have as many as 5,000 living members, and no other population in the world offers a sufficiently large number of mutation carriers close to the age of potential disease onset for a study to determine whether a prevention treatment may work. This effort by Genentech thus represents an application of a rare disease strategy to AD.

It is also possible, however that drugs that work by lowering levels of Aβ will not be efficacious in treating AD, even if administered early in the disease process. This may be true despite the findings of the new genetic study by the deCode Genetics group. For example, in their Nature News and Views article, Drs. De Strooper and Voet remind us that if the A673T mutation indeed works via lowering of Aβ levels, it works via lifelong lowering of Aβ, not lowering of Aβ in patients who already have AD, as in all clinical trials so far of anti-Aβ antibodies. (Even Genentech’s Colombian trial may involve lowering of Aβ levels relatively late in the course of exposure of patients to a disease process that will result in AD.)

Moreover, as these authors speculate on the basis of work on another mutation at the same site in the APP protein, it is possible that the protective effect of the A673T mutation may be due to changing the aggregation properties of Aβ peptides, resulting in a less-toxic form of Aβ. If true, this would mean that the protective effect of the A673T mutation is due to qualitative, rather than quantitative changes in Aβ. In that case, the finding of protection from AD by the A673T mutation might not be as predictive of the efficacy of such Aβ-lowering treatments as the use of anti-Aβ MAb drugs as drug developers might like.
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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Cancer immunotherapy: The star of the 2012 ASCO Annual Meeting

 


The American Society of Clinical Oncology (ASCO) held its 2012 Annual Meeting on June 1-5, 2012. Arguably the highlight of the meeting was the June 2, 2012 presentation by Bristol-Myers Squibb (BMS) on its Phase 1 immunotherapeutic, anti-PD-1 (BMS-936558). The results of this study were also published ahead of print on June 2, in the online version of the New England Journal of MedicineNature published a “News in Focus” article on the same subject by Nature staff writer Erika Check Hayden in its 6 June issue.

BMS acquired its anti-PD-1 MAb product BMS-936558 (MDX-1106) via its 2009 acquisition of Medarex. This is the same way in which BMS acquired its now-marketed immunotherapy, ipilimumab (Yervoy), which was approved by the FDA in March 2011. Both BMS-936558 and ipilimumab are monoclonal antibodies (MAbs). Ono Pharmaceuticals has been a partner in the development of anti-PD-1 MAb since its original collaboration with Medarex; Ono retains the right to exclusively develop and market the agent (which is also designated as ONO-4538) in Japan, Korea and Taiwan.

PD-1 (“programmed cell death-1”) is a receptor on the surface of activated T lymphocytes of the immune system. PD-1 is a member of the CD28/CTLA4 family of T cell regulators. Like CTLA4, the target of ipilimumab, PD-1 is a negative regulator of T-cell receptor signals. When a protein on the surface of some tumor cells, known as PD-1 ligand (PD-L1), binds to PD-1 on T cells that recognize antigens on these tumors cells, this results in the blockage of the ability of the T cells to carry out an anti-tumor immune response. Anti-PD-1 MAb binds to PD-1 on T cells, thus preventing PD-L1 on tumor cells from binding to the PD-1 and initiating an inhibitory signal. Anti-tumor T cells are then free to initiate immune responses against the tumor cells. This mechanism of action is completely analogous to that of ipilimumab, which binds to CTLA4 and thus prevents negative signaling from that molecule.

Phase 1 clinical study of Medarex/BMS’s anti-PD-1

The Phase 1 clinical study was carried out by a multi-institution team of investigators, led by Suzanne L. Topalian, M.D. (Johns Hopkins University School of Medicine, Baltimore, MD.) The researchers enrolled patients with advanced melanoma, non-small-cell lung cancer (NSCLC), prostate cancer, renal cell cancer (RCC), or colorectal cancer. Patients received anti-PD-1 at a dose between 0.1 and 10.0 milligrams per kilogram of body weight every two weeks. Tumor response was determined after each 8-week treatment cycle. Patients received up to 12 cycles of treatment until either unacceptable adverse events, disease progression or a complete response occurred. A total of 296 patients received treatment through February 24, 2012.

Among the 236 patients in whom tumor responses could be evaluated, objective responses were observed in patients with NSCLC, melanoma, or RCC. Cumulative response rates (among patients treated with all doses of anti-PD-1) were 18% among patients with NSCLC, 28% among patients with melanoma, and 27% among patients with RCC.  These responses were durable–20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. Anti–PD-1 produced objective responses in approximately one in four to one in five patients with NSCLC, melanoma, or RCC.

In addition to patients with objective responses, other patients treated with anti-PD-1 exhibited stable disease lasting 24 weeks or more–5 patients (7%) with NSCLC, 6 patients (6%) with melanoma, and 9 patients (27%) with RCC.

Significant drug-related adverse effects were seen in 11% of the patients, including three deaths due to pulmonary toxicity. In most cases, adverse effects were reversible, and the observed adverse-event profile does not appear to preclude the use of the drug. A maximum tolerated dose was not reached in this study.

The exciting finding of this study is that anti-PD-1 produced durable responses not only in melanoma and RCC (the two types of cancer that are deemed to be “immunogenic”), but also in NSCLC, a much more common cancer that kills more people per year than any other cancer. Moreover, response rates with anti-PD-1 were much higher that those achieved with the other recently approved immunotherapeutics. In the Phase 3 clinical trial of ipilimumab that led to its approval, this drug gave response rates of 11% in melanoma patients. The other recently approved immunotherapeutic, the prostate cancer-specific dendritic cell vaccine Sipuleucel-T (Dendreon’s Provenge, APC8015), gives very low response rates and no complete responses. According to Antoni Ribas (Jonsson Comp­rehensive Cancer Center, University of California, Los Angeles CA) as quoted Ms. Hayden’s Nature “News in Focus” review, if an immunotherapy “breaks the 10% ceiling” as did anti-PD-1, it becomes “even more important and clinically relevant”.

Despite the exciting efficacy results with anti-PD-1, and despite the fact that it was deemed that the adverse-event profile did not appear to preclude the use of the drug, researchers would still like to get away from the serious adverse effects (including three deaths) seen with anti-PD-1. As with other immunotherapeutics (e.g., ipilimumab), researchers hypothesize that anti-PD-1’s serious adverse effects were due to autoimmune responses.

Phase 1 clinical study of Medarex/BMS’ anti-PD-L1

A potential way of achieving similar efficacy to anti-PD-1 with an improved safety profile is provided by another Phase 1 immunotherapeutic,  anti-PD-L1. Anti-PD-L1 MAb drugs are being developed by Medarex/BMS, Roche/Genentech, and other companies. As mentioned earlier, PD-L1 is the binding partner of PD-1 that is expressed on some tumor cells. As quoted in the Nature “News in Focus” review, Ira Mellman (vice-president of research oncology at Genentech), believes that anti-PD-L1 might have fewer adverse effects than anti-PD-1. That is because anti-PD-L1 would target tumor cells while leaving T cells free to participate in immune networks that work to prevent autoimmune reactions.

The results of a Phase 1 clinical study of BMS/Medarex’ anti-PD-L1 (also known as MDX-1105) were also published ahead of print in the online version of the New England Journal of Medicine on June 2, 2012; this was a “companion study” to the Phase 1 study of anti-PD-1. This study was also carried out by a multi-institution team of investigators, led by Julie R. Brahmer, M.D. (Johns Hopkins University School of Medicine, Baltimore, MD.); Dr. Topalian, among other investigators on the anti-PD-1 trial, also participated in the study.

This Phase 1 trial was a dose escalation study that was carried out via a similar protocol to the anti-PD-1 trial discussed earlier. As of February 24, 2012, a total of 207 patients — 75 with NSCLC, 55 with melanoma, 18 with colorectal cancer, 17 with RCC, 17 with ovarian cancer, 14 with pancreatic cancer, 7 with gastric cancer, and 4 with breast cancer — had received anti–PD-L1 antibody, for a median duration of 12 weeks. Among patients with an evaluable response, an objective response (i.e., a complete or partial response) was seen in 17% of patients with melanoma, 12% of patients with RCC, 10% of patients with NSCLC, and 6% of patients with ovarian cancer. Responses lasted for 1 year or more in 8 of 16 patients with at least 1 year of follow-up. Prolonged disease stabilization was seen in 12-41% of patients with advanced cancers, including NSCLC, melanoma, and RCC.

Significant drug-related adverse effects were seen in 9% of patients.

Although the two agents were not compared directly in a randomized trial, the frequency of objective responses for anti–PD-L1 MAb appears to be somewhat lower than that observed for anti–PD-1 MAb in initial Phase 1 trials; the frequency and severity of significant drug-related adverse events also appears to be lower. However, whether these differences will hold up in Phase 2 and 3 clinical trials remains to be determined. The clinically appropriate dose of anti–PD-L1 will also require further definition later studies. Nevertheless, the Phase 1 trial showed that anti-PD-L1 MAb induced durable tumor regression (objective response rate of 6-17%) and prolonged disease stabilization (rate of 12-41% at 24 weeks) in patients with select advanced cancers, including NSCLC, a tumor type that had been deemed to be “non-immunogenic”. This is essentially the same result that was observed for anti-PD-1MAb.

A predictive biomarker for treatment with anti-PD-1?

As with other modes of cancer therapy, it would be very useful to have mechanism-based predictive biomarkers to identify appropriate candidates for treatment with anti-PD-1 or anti-PD-L1 immunotherapy. The findings of the Phase 1 anti-PD-1 study suggest that PD-L1 expression in tumors is a candidate biomarker that warrants further evaluation for use in selecting patients for immunotherapy with anti–PD-1 MAb. The researchers found that 36% of patients with PD-L1–positive tumors achieved an objective response, while no patients with PD-L1–negative tumors achieved such a response. These results suggest that PD-L1 expression on the surface of tumor cells in pre-treatment tumor specimens may be associated with an objective response. However, further studies will be necessary to define the role of PD-L1 as a predictive biomarker of response to anti–PD-1 therapy. Similarly, it appears reasonable that tumor expression of PD-L1 may be a predictive biomarker of response to anti-PD-L1 therapy. However, this hypothesis must also be tested in further clinical studies.

Further studies of anti-PD-1 MAb

Two studies of BMS-936558/MDX-1106 anti–PD-1 MAb, both in advanced/metastatic clear-cell RCC, are now recruiting patients. One trial is a Phase 1 biomarker study involving immunologic and tumor marker correlates of efficacy (progression-free survival and tumor response). The other trial is a Phase 2 efficacy (progression-free survival and tumor response) study; this is a dose ranging study that is designed to determine if a dose response exists. Phase 3 studies of BMS-936558/MDX-1106 anti–PD-1 MAb for the treatment of non–small-cell lung cancer, melanoma, and renal-cell cancer are also being planned.

Conclusions

The exciting results of the studies with BMS’ anti-PD-1 and anti-PD-L1 have only been in Phase 1 studies. Thus caution is advisable in interpreting these results, pending the results of further clinical studies. Nevertheless, these results, together with the recent approval of ipilimumab (Medarex/Bristol-Myers Squibb’s Yervoy) and of Sipuleucel-T (Dendreon’s Provenge), indicate that cancer immunotherapy, a field that not so long ago was regarded as an impractical dream, is very much alive and well. In addition to clinical development and approval of immunotherapeutic agents, exciting basic and drug discovery research in this field is ongoing. This was recognized by the awarding of the 2011 Nobel Prize in Physiology or Medicine for research with profound implications for the development of cancer immunotherapies.

The Biopharmconsortium Blog has been covering new developments in cancer immunotherapy since the spring of 2011. Our earlier articles on this subject (with links) are listed in our December 31, 2011 article, entitled “Read the cancer immunotherapy review in the 22 December 2011 issue of Nature!”

Cancer immunotherapy represents one of several “scientifically premature” or “frontier science” areas discussed in this blog that are providing new opportunities for drug discovery and development–for young entrepreneurial biotech start-ups and for more established biotechnology and pharmaceutical companies. Corporate strategists would do well to explore such areas for potential new R&D programs for their companies.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Co-clinical mouse/human trials for cancer continue to advance

 

RAS/BRAF/PI3K pathways. Source: Source BioScience

Two previous articles on this blog have included discussions of the “co-clinical mouse/human trial” strategy for improving mouse models of human cancer, and simultaneously improving human clinical trials of drugs for these cancers. Now comes an article on the use of a co-clinical trial strategy in personalized treatment of non-small cell lung cancer (NSCLC) in the 29 March 2012 issue of Nature. In the same issue of Nature is a News and Views article by Genentech’s Leisa Johnson Ph.D. that provides a minireview of the research article.

As we discussed in our April 15, 2010 article on this blog, the co-clinical trial strategy has been developed by Pier Paolo Pandolfi, MD, PhD (Director, Cancer and Genetics Program, Beth Israel-Deaconess Medical Center Cancer Center and the Dana-Farber/Harvard Cancer Center) and his colleagues.

As discussed in that article, these researchers constructed genetically engineered transgenic mouse strains that have genetic changes that mimic those found in human cancers. These mouse models spontaneous develop cancers that resemble the corresponding human cancers. In Dr. Pandolfi’s  ongoing co-clinical mouse/human trial project, researchers simultaneously treat a genetically engineered mouse model and patients with tumors that exhibit the same set of genetic changes with the same experimental targeted drugs. The goal of this two-year project is to determine to what extent the mouse models are predictive of patient response to therapeutic agents, and of tumor progression and survival. The studies may thus result in validated mouse models that are more predictive of drug efficacy than the currently standard xenograft models.

The human clinical trials being “shadowed” by simultaneous studies in mice included Phase 3 trials of several targeted therapies for lung and prostate cancer. Xenograft models in which tumor tissue from the patients had been transplanted into immunosuppressed mice were also being tested in parallel with the genetically engineered mouse models. This project represents the most rigorous test to date of how well genetically engineered mouse models of cancer can predict clinical outcomes.

Our October 28, 2011 blog article, which is mainly a review of a 29 September 2011 Nature article by Nature writer Heidi Ledford, Ph.D., focuses on ways to fix the clinical trial system. Our article includes a discussion of a co-clinical trial published in January 2011. This trial utilized two genetically-engineered PDGF (platelet-derived growth factor)-driven mouse models of the brain tumor glioblastoma multiforme (GBM), one of which had an intact PTEN gene and the other of which was PTEN deficient. In this trial, researchers tested the Akt inhibitor perifosine (Keryx Biopharmaceuticals, an alkylphospholipid) and the mTOR inhibitor CCI-779 (temsirolimus; Pfizer’s Torisel), both alone and in combination, in vitro and in vivo. The drugs and drug combinations were tested in cultured primary glioma cell cultures derived from the PTEN-null and PTEN-intact mouse PDGF-driven GBM models, and in the animal models themselves.

The studies showed that both in vitro and in vivo, the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null cells in animals was achieved by using both inhibitors in combination.  In vivo, the decreased Akt and mTOR signaling seen in mice treated with the combination therapy correlated with decreased tumor cell proliferation and increased cell death; these changes were independent of PTEN status. The co-clinical animal study also suggested new ways of screening GBM patients for inclusion in clinical trials of treatment with perifosine and/or CCI-779.

The new co-clinical trial reported in the March 2012 issue of Nature

The March 2012 Nature report describes research carried out by a large, multi-institution academic consortium, which included Dr. Pandolfi. It focuses on strategies for treatment of patients with non-small-cell lung cancer (NSCLC) with activating mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog). These mutations occur in 20–30% of NSCLC cases, and patients whose tumors carry KRAS driver mutations have a poor prognosis. Moreover, KRAS is a “hard” or “undruggable” target, and no researchers have thus been able to discover inhibitors of oncogenic KRAS.

Because of the intractability of oncogenic KRAS as a target, researchers have been attempting to develop combination therapies for mutant-KRAS tumors (including, for example, colorectal cancers as well as NSCLCs) that address downstream pathways controlled by KRAS. We discussed examples of these strategies in our book-length report Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, published by Cambridge Healthtech Institute/Insight Pharma Reports in 2011. Strategies discussed in that report are based on the finding that KRAS controls signal transduction via two key pathways: the B-Raf-MEK-ERK pathway and the PI3K-Akt pathway. This is illustrated in the figure at the top of this article. As discussed in our 2011 report, researchers are attempting to develop treatments of mutant-KRAS tumors that involve combination therapies with an inhibitor of the mitogen-activated protein kinase (MEK) together with an inhibitor of phosphatidylinositol 3-kinase (PI3K). Researchers are also attempting to develop combination therapies of MEK inhibitors with standard cytotoxic chemotherapies, which if successful will avoid having to use combinations of two expensive targeted therapies.

In the co-clinical trial that is the focus of the 29 March 2012 Nature research report and News and Views commentary, researchers developed a genetically-engineered mouse model to study treatment of mutant-KRAS NSCLCs with either the antimitotic chemotherapy drug docetaxel alone, or docetaxel in combination with the MEK kinase inhibitor selumetinib (AZD6244, AstraZeneca). In the parallel human clinical trial, researchers are also studying treatment of patients with mutant-KRAS NSCLC with docetaxel alone or docetaxel plus selumetinib. (There is no treatment arm in the human clinical trial in which patients are treated with selumetinib alone, since selumetinib monotherapy of NSCLC patients had shown no efficacy in a previous Phase 2 study; this was confirmed in mouse model studies.)

In humans with mutant-KRAS NSCLC, many tumors with mutations in KRAS have concomitant genetic alterations in other genes that may affect response to therapy. Therefore, the co-clinical trial researchers wished to design mouse models with lung tumors with either Kras mutations alone or with mutations in both Kras and another gene that is often concomitantly mutated in mutant-KRAS NSCLCs in humans. The researchers therefore constructed mouse models with cancers bearing the activating Kras(G12D) mutation, either alone or together with an inactivating mutation in either p53 or Lkb1. The researchers achieved this via a conditional mutation system using nasal instillation of specifically genetically-engineered adenoviruses. As result, a small percentage of lung epithelial cells harbored these mutations. It is from these cells that the NSCLC-like tumors arose, analogous to the clonal origin of sporadic lung tumors in humans.

Of the two tumor suppressor genes that are frequently mutated in human mutant-KRAS NSCLCs and that were modeled by the co-clinical trial researchers, p53, often called the “guardian of the genome”, is familiar to most of you. The other gene, Lkb1 [liver kinase B1, also known as serine/threonine kinase 11 (STK11)], was discussed in an earlier article on the Biopharmconsortium Blog, entitled “The great metformin mystery–genomics, diabetes, and cancer.”

LKB1 (whether in regulation of gluconeogenesis in the liver or in its role as a tumor suppressor) acts by activation of AMPK (AMP-activated kinase, a sensor of intracellular energy status.) In lung cancer (as shown by the same group that performed the 2012 co-clinical trial), LKB1 acts to modulate lung cancer differentiation and metastasis.  Germline mutations in LKB1 are associated with the familial disease Peutz-Jegher syndrome, in which patients develop benign polyps in the gastrointestinal tract. Studying a mouse model of mutant-LKB1 Peutz-Jeger syndrome, Reuben J. Shaw (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA, who was prominently mentioned in our “great metformin mystery” article) and his colleagues showed that the LKB1-AMPK pathway downregulates the mTOR pathway–specifically the rapamycin-sensitive mTOR complex 1 (mTORC1) and its downstream effector hypoxia-inducible factor-1α (HIF-1α). HIF-1α expression in turn upregulates the expression of its downstream effectors hexokinase II and glucose transporter 1 (GLUT1), which are involved in cellular utilization of glucose. LKB1-deficient polyps in this mouse model thus show increased expression of hexokinase II and GLUT1, resulting in dramatically increased glucose utilization.

In the new co-clinical trial, genetically-engineered mice that showed established lung tumors [as determined via magnetic resonance imaging (MRI)] were randomized to receive either docetaxel, selumetinib, or a combination of the two drugs. For tumors with only a Kras mutation, treatment with docetaxel alone resulted in a modest rate of response, with 30% of mice showing a partial response. Mice that bore mutant-Kras tumors that also had mutations in either p53 or Lkb1 had much lower rates of response to docetaxel monotherapy (5% and 0%, respectively), and more of these mice showed progressive disease on MRI or died of their disease. Of mice treated with the docetaxel/selumetinib combination, those with single-mutant Kras tumors showed a 92% overall response rate, and those with mutant Kras/p53 tumors showed a 61% overall response rate. However, mice with mutant Kras/Lkb1 cancers showed only a modest response to the docetaxel/selumetinib combination; 33% of mice achieved a partial response. The difference in response rate of mice with Kras/Lkb1 tumors to docetaxel/selumetinib compared to the other two genotypes was found to be statistically significant.

Using the genetically-engineered NSCLC mouse model in biomarker development

In human patients in clinical trials or in treatment for their cancers, performing repeated biopsies to monitor treatment is difficult. The co-clinical trial researchers therefore wished to develop less invasive means of monitoring both co-clinical and clinical trials of docetaxel/selumetinib treatment of NSCLC. They therefore tested the use of positron emission tomography (PET) with 18F-fluoro-2-deoxyglucose (FDG-PET) as an indicator of early response to therapy that could be used in the clinic.  Prior to its radioactive decay (109.8 minute half -life), 18F-FDG is a nonmetabolizable glucose analogue that moves into cells that is preferentially taken up by high-glucose utilizing cells. The researchers found that both Kras/p53 and Kras/Lkb1 tumors showed higher FDG uptake in vivo in the mouse model than did single-mutant Kras tumors. As expected from the earlier study, GLUT1 expression was elevated in Kras/Lkb1 mutant tumors. In human patients, pre-treatment, mutant KRAS/LKB1 tumors also showed a higher FDG uptake that did KRAS tumors negative for LKB1.

Treatment of the mice with docetaxel alone gave no significant changes in FDG uptake in Kras, Kras/p53, or Kras/Lkb1 tumors in vivo. However, within 24 hours of the first dosing of docetaxel/selumetinib, FDG uptake was markedly inhibited in Kras and Kras/p53 tumors. In contrast, treatment of mice with Kras/Lkb1 mutant tumors gave no appreciable decrease in FDG uptake in these tumors. These results show that early changes in tumor metabolism, as assessed by FDG-PET, predict antitumor efficacy of docetaxel/selumetinib treatment.

The FDG-PET study in this mouse model supports the use of this imaging method as a biomarker to monitor the course of treatment in humans.

Cellular signaling in mutant Kras, Kras/p53, and Kras/Lkb1 tumors

The researchers assessed activation of relevant intracellular pathways in tumors in treated and untreated mice with mutant Kras, Kras/p53, and Kras/Lkb1 lung cancers. They performed these studies using two different methods–immunostaining of cancer nodules for phosphorylated ERK, and immunoblotting of tumor lysates. In untreated mice, Kras/Lkb1 tumors show less activation of ERK than do Kras and Kras/p53 tumors, with Kras/p53 tumors showing the greatest amount of activation of the MEK-ERK pathway. Docetaxel had no discernible effect on signaling via the MEK-ERK pathway. Selumetinib alone resulted in decreased ERK activation in Kras and Kras/p53 tumors, but there was still residual activity. The docetaxel/selumetinib combination, however, was more effective in eliminating ERK activation. Pharmacokinetic studies indicated that selumetinib levels were higher in both serum and tumors of mice treated with docetaxel/selumetinib that in those treated with selumetinib alone; this might account for the more potent suppression of MEK-ERK signaling by the combination therapy as compared to selumetinib monotherapy. The researchers studied MEK-ERK activation (as determined by phospho-ERK staining) in  a set of 57 human NSCLC tumors with known RAS, p53 and LKB1 mutation status. As with the tumors in the mouse model, of seven patients whose tumors harbored the KRAS activating mutation, the three patients with concurrent p53 mutations showed higher levels of ERK activation.

The decreased activation of ERK in Kras/Lkb1 tumors suggested that these tumors utilize other pathways to drive their proliferation. On the basis of their prior studies of signal transduction in mutant-Lkb1 lung tumors, the researchers focused on AKT and SRC. Immunoblotting studies showed that Kras/Lkb1 mutant tumors had elevated activation of both AKT and SRC. As one can see from the figure at the top of this article, AKT is a downstream effector of PI3K; since the PI3K/AKT pathway regulates expression of GLUT1 and hexokinase, increased activation of the PI3K/AKT pathway is consistent with the increased uptake of FDG of mutant Kras/Lkb1 tumors. In the figure, SRC (which is not shown) represents one of the major “other effectors” controlled by RAS. These results indicate that concomitant mutation of Lkb1 in mutant-Kras NSCLCs may shift the signaling pathways that drive tumor proliferation from MEK-ERK to PI3K/AKT and/or SRC. This shift would result in primarily resistance of Kras/Lkb1 tumors to treatment with docetaxel/selumetinib.

Long-term benefits of treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel/selumetinib as opposed to docetaxel monotherapy

The researchers studied long-term treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel monotherapy versus docetaxel/selumetinib. In mice with mutant-Kras tumors, treatment with docetaxel monotherapy gave stable disease for several weeks, while docetaxel/selumetinib treatment resulted in tumor regression and slower regrowth of tumors. The addition of selumetinib to docetaxel significantly prolonged progression-free survival in these mice. In mice with Kras/p53 tumors, treatment with docetaxel alone resulted in progressive disease, but docetaxel/selumetinib treatment resulted in initial disease regression followed by progression, resulting in prolonged progression-free survival. These results indicate that treatment with combination therapy as opposed to docetaxel alone results in improved progression-free survival, but not cure, in mice with Kras– and Kras/p53-mutant tumors.

The researchers also investigated mechanisms of acquired tumor resistance in mice with mutant-Kras and Kras/p53 tumors, which had been treated long-term with docetaxel/selumetinib. In moribund animals that had received this treatment, all tumor nodules examined showed a recurrence of ERK phosphorylation. This suggested that acquired resistance could be at least in part due to reactivation of MEK–ERK signaling despite ongoing treatment with selumetinib. Evaluation of resistant tumor nodules suggested that more than one mechanism for pathway reactivation was occurring; study of these mechanisms is ongoing.

Conclusions of the new co-clinical study

The results of this co-clinical study predict that docetaxel/selumetinib combination therapy will be more effective than docetaxel monotherapy in several sub-classes of mutant-KRAS NSCLC. This prediction is consistent with the early results of a Phase 2 clinical trial of these two drug combinations in second-line treatment of patients with KRAS-mutant NSCLC.

However, the co-clinical trial also predicts that concurrent mutation of LKB1 in mutant-KRAS  tumors will result in primary resistance to docetaxel/selumetinib combination therapy, perhaps via activation of parallel signaling pathways such as AKT and SRC. Since LKB1 status is not being prospectively assessed in the ongoing human clinical trial, the presence of patients with cancers having concurrent LKB1 mutations may diminish the differences between treatment arms based solely on KRAS status. The results of the co-clinical trial suggests that researchers perform retrospective analysis of p53 and LKB1 status in samples from the concurrent human clinical trial. Future clinical trials should then be designed that involve prospective analysis to ensure sufficient enrollment of patients with all three genotypes to enable sufficiently powered sub-group analyses.

Although the results of the co-clinical trial indicate that patients with mutant KRAS/LBK1 tumors be excluded from trials of docetaxel/selumetinib treatment, the research group that has been conducting the co-clinical trial has also been conducting studies that may lead to treatment strategies for KRAS/LBK1 tumors.

The co-clinical trial also allowed researchers to design and validate biomarker strategies, specifically the potential use of the less-invasive FDG-PET to predict efficacy and to monitor treatment. The co-clinical animal-model study also enabled the discovery of mechanisms of both primary and acquired resistance that might benefit future clinical trials and discovery/development of drugs. (The studies on acquired resistance are in a early stage and are ongoing). Any mechanisms of acquired resistance discovered in co-clinical studies should be confirmed in human clinical trials by examining biopsy samples from patients who relapse on therapy. The ability to assess mechanisms of resistance in preclinical or co-clinical animal studies may enable researchers to design rational drug combination strategies that can be implemented in future clinical studies.

The results of the new co-clinical trial strengthens the contention that co-clinical trials in genetically-engineered mice can provide data that can predict the outcome of parallel human clinical trials. Co-clinical trials can also be used to generate new hypotheses for use in analyzing concurrent human trials, and for design of future clinical studies. Moreover, co-clinical trials can result in the validation of improved animal models for human cancers, which can be used in research and preclinical testing of oncology agents, and in validation of biomarkers for clinical studies in oncology. Given the inadequacy of “standard” xenograft models, which is a major factor in the high attrition rate of pipeline oncology drugs, the availability of validated genetically-engineered animal models may be expected to enable improved oncology drug development.

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